Pyrrolidine Alkaloids from Mangrove Fungus Penicillium sp. DM27 Enhance L6 Cell Glucose Uptake

Abstract

Ten previously undescribed pyrrolidine alkaloids, namely penicipyrrolidines O–X (1–10), were isolated from the mangrove-derived fungus Penicillium sp. DM27, along with five known compounds (11–15). Their structures were determined by comprehensive analysis of HRESIMS and NMR spectroscopic data, and the absolute configurations were established based on biosynthetic considerations and TDDFT-ECD calculations. All isolates were evaluated for their glucose uptake capacity. Notably, penicipyrrolidine P (2) significantly enhanced cellular glucose uptake in L6 myotubes by 3.83-fold, demonstrating activity comparable to that of metformin, whereas penicipyrrolidines Q and R (3 and 4) showed relatively weaker effects.

1. Introduction

Mangrove-associated fungi are a prolific source of diverse bioactive metabolites. Endophytic fungi, once poorly documented in mangrove roots [1], have emerged as promising sources of complex compounds with antimicrobial, antioxidant, and enzymatic activities [2,3,4,5,6,7,8]. Fungi belonging to the genus Penicillium are especially recognized as producers of structurally unique and biologically active metabolites [9]. Notably, natural products containing pyrrolidine core exhibit a broad spectrum of pharmacological activities, such as antimicrobial, anticancer, and enzyme inhibitory effects [10]. Nevertheless, only a limited number of pyrrolidine derivatives containing the distinctive 2-methyl-1-(pyrrolidinyl)-decanone scaffold have been reported from marine-derived Penicillium species [11,12,13,14,15].

Our prior research on the mangrove-derived fungus Penicillium sp. DM27 led to the isolation and characterization of nineteen novel pyrrolidine alkaloids (penicipyrrolidines A–N) and three pyrrolizidinone alkaloids, penicipyrrolizidinones A–C, with unprecedented skeletons [16,17,18]. While penicipyrrolidine K demonstrated potent anti-fibrotic activity by targeting ADAM17, and two compounds exhibited moderate cytotoxicity, the overall exploration of biological activities for these structurally diverse pyrrolidine derivatives from the strain DM27 remains limited [16,17].

Motivated by the structural richness and untapped bioactivity potential of pyrrolidine alkaloids from the title fungus, we turned our attention to diabetes mellitus, a widespread metabolic disorder marked by progressive β-cell dysfunction and hyperglycemia [19,20]. Through large-scale fermentation of Penicillium sp. DM27, ten new pyrrolidine alkaloids, designated penicipyrrolidines O–X (1–10), along with five known analogues (11–15), were obtained (Figure 1). Notably, penicipyrrolidines P–R (2–4) exhibited stimulatory effects on glucose uptake, highlighting their potential as antidiabetic agents and prompting further investigation.

Figure 1. Structures of compounds 1–15.

2. Results and Discussion

2.1. Structural Analysis

Penicipyrrolidine O (1) was isolated as a colorless oil, and assigned a molecular formula of C₁₃H₂₁NO₂ (four degrees of unsaturation) based on HRESIMS ions at m/z [M + Na]⁺ 246.1463 and ¹³C NMR data (Table 1). The ¹H NMR spectrum of 1 exhibited four olefinic proton signals (δ_H 6.96, 6.12, 5.46, 5.42) and one methyl at δ_H 1.65. The ¹³C NMR and HSQC of 1 showed an amide carbonyl (δ_C 167.6), four olefinic carbon signals (δ_C 147.1, 129.9, 126.2, 121.6), one oxymethylene (δ_C 67.9), one methine (δ_C 61.6), five methylenes (δ_C 48.2, 32.7, 31.4, 28.5, 24.6), and one methyl (δ_C 18.1). Four degrees of unsaturation were calculated from its molecular formula, the carbonyl and the two double bonds accounted for three, which suggested a ring in 1. Comprehensive analysis of the NMR data (Table 1), particularly the characteristic proton resonances at CH-2, CH₂-3, CH₂-4, CH₂-5 and CH₂-6, combined with COSY correlations of C-2(–C-6)–C-3–C-4–C-5, established 1 as a pyrrolidine alkaloid featuring a pyrrolidin-2-ylmethanol substituent like scalusamide A (12) [11]. This assignment was confirmed by HMBC correlations of H-3 with C-6, H-4 with C-2 and C-3, and H-5 with C-2 and C-3 (Figure 2). The COSY spectrum established a continuous spin system, CH-2′/CH-3′/CH₂-4′/CH₂-5′/CH-6′/CH-7′/CH₃-8′, which was further confirmed and defined as a hepta-1,5-diene chain by the key HMBC correlations from H-2′ to C-4′, H-7′ to C-5′, and from both H-4′ and H-8′ to C-6′. The HMBC correlations of H-2′ and H-3′ with C-1′ implied that the amide carbonyl was connected to C-2′. Comparative analysis of scalusamides A–C (12–14) demonstrated that the pyrrolidin-2-ylmethanol substituent is covalently linked to the hepta-1,5-diene substituent via the carbonyl group C-2′. Therefore, the planar structure of 1 was determined as shown [11]. Notably, compound 1 is the first pyrrolidine alkaloid featuring an eight-carbon chain attached to the nitrogen atom of pyrrolidin-2-ylmethanol substituent.

Table 1. ¹H and ¹³C NMR spectroscopic data for compounds 1–4 in CDCl₃ (δ in ppm, J in Hz).

No.	1 a		2 b		3 b		4 b
	δC	δH	δC	δH	δC	δH	δC	δH
2	61.6	4.28, m	128.4	6.50, m	128.7	6.54, m	128.4	6.56, d (4.5)
3	28.5	2.05, m; 1.59, m	112.9	5.26, m	113.7	5.29, m	113.5	5.29, m
4	24.6	1.96, dt (13.2, 6.5) 1.88, dt (12.5, 6.5)	28.2	2.60, m	28.6	2.66, m	28.3	2.78, t (9.1)
5	48.2	3.63, m; 3.55, m	44.9	3.80, m	46.0	3.86, t (9.3)	45.6	3.87, t (9.0)
6	67.9	3.68, m; 3.61, m
1′	167.6		172.5		165.7		165.7
2′	121.6	6.12, dt (15.0,1.7)	42.4	2.56, m	53.1	3.58, q (7.0)	53.2	3.58, q (7.0)
3′	147.1	6.96, dt (15.0, 7.0)	74.0	3.59, m	207.5		206.1
4′	32.7	2.28, q (7.0)	35.3	1.45, m	39.7	2.54, m; 2.47, m	39.1	2.60, m; 2.53, m
5′	31.4	2.15, dt (8.7, 7.0)	25.5	1.45, m; 1.32, m	23.8	1.60, m	26.1	2.30, m
6′	129.9	5.42, m	29.6	1.34, m	25.5	1.42, m; 1.32, m	130.0	5.59, m
7′	126.2	5.46, m	32.6	1.94, m	37.0	1.43, m	134.1	5.44, dd (15.7, 6.5)
8′	18.1	1.65, d (4.9)	131.4	5.37, m	73.4	3.48, m	74.3	3.93, m
9′			124.8	5.37, m	30.6	1.45, m	30.3	1.51, m
10′			18.0	1.60, d (3.9)	10.4	0.92, t (7.4)	9.9	0.87, t (7.4)
11′			15.1	1.21, d (7.1)	13.6	1.37, d (7.0)	13.2	1.37, d (7.1)

^a Spectra were recorded at 400 MHz for ¹H NMR and at 151 MHz for ¹³C NMR. ^b Spectra were recorded at 400 MHz for ¹H NMR and at 101 MHz for ¹³C NMR.

Figure 2. Key ¹H-¹H COSY (red bold) and HMBC (blue arrows) correlations of compounds 1–10.

The trans-configuration of the C-2′–C-3′ double bond in 1 was unambiguously confirmed by the observed vicinal coupling constant (³J_{H-2′/H-3′} = 15.0 Hz). The E configuration of the C-6′–C-7′ double bond was assigned based on the upfield chemical shift of C-8′ (δ_C 18.1, <20.0 ppm), by analogy to the established correlation for the C-13–C-14 bond in scalusamide A (12) (δ_C 17.7 for C-15) [11,21]. Based on the biosynthetic considerations, the pyrrolidin-5-ylmethanol moiety was suggested to originate from the reduction of R-proline [11]. Thus, the absolute configuration of C-2 in 1 was determined to be R.

Penicipyrrolidine P (2) was obtained as a white amorphous powder, the molecular formula was inferred to be C₁₅H₂₅NO₂ from its HRESIMS ions at m/z [M + H]⁺ 252.1953 (calcd for 252.1964) and ¹³C NMR data (Table 1), indicating four degrees of unsaturation. The ¹H NMR spectrum of 2 exhibited four olefinic protons (δ_H 6.50, 5.37, 5.37, 5.26) and two methyls at δ_H 1.60 and 1.21. The ¹³C NMR and HSQC of 2 showed two sets of signals for an amide carbonyl (δ_C 172.4 and 172.5), four olefinic carbon signals (δ_C 131.4, 128.4, 124.8, 112.9 and 131.4, 128.8, 124.7, 111.7), one oxymethine (δ_C 74.0 and 74.1), one methine (δ_C 42.4 and 42.7), six methylenes (δ_C 44.9, 35.3, 32.6, 29.6, 28.2, 25.5 and 45.6, 35.3, 32.5, 30.0, 28.1, 25.4), and two methyls (δ_C 18.0, 15.1 and 17.9, 14.9). The carbonyl group and two double bonds represent three, suggesting a ring in 2. Considering the rotameric phenomenon of 2, compound 2 was considered as a rotational isomer for the amide carbonyl linked with an asymmetric pyrrolidine moiety [11].

The ¹H-¹H COSY NMR data showed two isolated proton spin systems of the 2-decene-8-hydroxy-9-yl fragment, and C-2–C-3–C-4–C-5 subunits of 2 (Figure 2). HMBC correlations from H-4 and H-5 to C-2 resulted the formation of a dihydropyrrole ring. The connectivity among the two isolated spin systems and the one carbonyl group (C-1′) was established by analysis of the HMBC spectrum. HMBC correlations from H-5, H-3′ and H₃-11′ to C-1′ led to form the planar structure of 2. After relentless structural search it was found that the planar structure of 2 was similar to that of previously reported N-(2-methyl-3-oxodec-8-enoyl)-2-pyrroline (15) [22]. The only difference observed was the reduction of the keto carbonyl group at C-3′ in 15 to a hydroxyl group in 2. In the ¹H NMR spectrum of 2, some signals are highly overlapped, rendering the absolute configuration of C-3′ challenging to ascertain through techniques such as the modified Mosher′s method (Figure S13).

Penicipyrrolidine Q (3) was isolated as a colorless oil, and assigned a molecular formula of C₁₅H₂₅NO₃ (four degrees of unsaturation) by HRESIMS 290.1718 [M + Na]⁺ (calcd for 290.1732) and ¹³C NMR data (Table 1). The planar structure of 3 was established by analysis of ¹H-¹H COSY and HMBC spectra. Detailed comparison of the NMR data of 3 with those of 15 revealed the same skeleton. The most significant difference was the disappearance of the C-8′–C-9′ double bond, which was replaced by a methylene group and an oxygenated methine group in 3, indicating that 3 is a hydration product of 15. The presence of the hydroxyl group at C-8′ was supported by the key HMBC correlations from H-6′ and H-10′ to it (Figure 2). Thus, the planar structure of 3 was established as shown.

As with 2, the assignment of the absolute configuration at C-3′ in 3 is challenging due to its rotameric nature. Although we attempted to determine the absolute configuration of the secondary hydroxyl group at C-8′ using the modified Mosher′s method, it was unsuccessful due to signal overlapping caused by rotational isomer.

Penicipyrrolidine R (4) was obtained as colorless oil and was inferred to have the molecular formula C₁₅H₂₃NO₃ from its protonated ion [M + Na]⁺ at m/z 288.1563 (calcd 288.1576 for C₁₅H₂₃NO₃), suggesting one more degree of unsaturation than that of 3. Detailed interpretation of the 1D and 2D NMR spectra (Table 1) of 4 revealed structural similarities to 3, suggesting they shared the same skeleton. The major difference was the presence of an additional double bond between C-6′ and C-7′ in 4. The foregoing information is also supported by the HMBC correlations from H-6′ (δ_H 5.59, m) and H-7′ (δ_H 5.44, dd, J = 15.7, 6.5 Hz) to the oxymethine at C-8′ (δ_C 74.2) and C-5′ (δ_C 26.0) (Figure 2). Thus, the planar structure of 4 was established as shown. The coupling constant ³J_{H-6′/H-7′} = 15.7 Hz suggested the E configuration in 4. Following a C₁₈ HPLC separation, 4 was separated into 4a and 4b. However, 4a and 4b were readily epimerized at C-2′ to each other within 48 h, a phenomenon previously observed in 12 [11]. The absolute configuration at C-8′ of 4 could not be determined due to rotational isomer, as well as the epimerization phenomenon [11].

The molecular formula of penicipyrrolidine S (5) was established as C₁₅H₂₅NO₃, indicating an identical molecular formula to that of 3. A detailed analysis of the NMR spectra (Table 2) suggested that 5 is a pyrrolidine alkaloid with the same skeleton as 15 [22]. Apart from the same fragment of N-(2-methyl-3-oxodec-8-enoyl)pyrrolidine, the most obvious difference was the signals for the C-2–C-3 double bond in 15 were absent, replaced by those for a methylene group and an oxygenated methine group (C-2). The ¹H-¹H COSY correlations of C-2–C-3–C-4–C-5 and HMBC correlations from H-5 and H-4 to C-2 (δ_C 89.7) supported the presence of a hydroxyl group at C-2 in 5 (Figure 2). Therefore, all of the atom connections in 5 were established as shown. In contrast to 2–4, rotational isomerism was not observed for 5, which can be attributed to the steric hindrance imposed by a bulky substituent at C-2. Attempts to assign the absolute configuration of 5 using the modified Mosher′s method were hampered by epimerization at C-2′ during the prolonged standing.

Table 2. ¹H and ¹³C NMR spectroscopic data for compounds 5–7 in CDCl₃ (δ in ppm, J in Hz).

No.	5 a		6 b,c				7 b
	δC	δH	δC	δH	δC	δH	δC	δH
1-NH								6.23, s
2	89.7	5.62, m	92.6	5.29, s	94.3	4.70, s	34.7	3.28, q (7.3)
3	29.8	1.97, m	73.2	4.20, m	73.3	4.37, m	14.8	1.12, d (7.3)
4	21.3	2.03, m; 1.91, m	31.1	2.27, m	30.0	2.19, m
5	45.8	3.56, m; 3.51, m	44.6	3.57, m	44.2	3.64, m
1′	170.1		171.4		172.2		169.5
2′	52.7	4.08, q (7.2)	53.0	3.49, m	53.0	3.57, m	54.4	3.41, q (7.2)
3′	208.6		206.8		207.5		210.6
4′	39.3	2.49, td (7.4, 1.1)	39.2	2.41, m	39.8	2.53, m	41.7	2.54, td (7.3, 3.7)
5′	23.1	1.53, p (7.4)	23.1	1.53, m	23.1	1.53, m	22.9	1.55, p (7.4)
6′	29.1	1.34, m	29.1	1.29, m	29.1	1.29, m	29.0	1.32, m
7′	32.5	1.98, m	32.4	1.94, m	32.4	1.94, m	32.4	1.97, m
8′	131.0	5.38, m	131.2	5.38, m	131.2	5.38, m	130.9	5.39, m
9′	125.3	5.40, m	125.3	5.39, m	125.3	5.39, m	125.2	5.39, m
10′	18.0	1.63, d (4.7)	18.1	1.62, d (3.6)	18.1	1.62, d (3.6)	18.0	1.63, d (4.9)
11′	14.0	1.41, d (7.1)	13.8	1.36, d (7.0)	14.2	1.41, d (7.1)	15.5	1.37, d (7.2)
2-OCH3			57.1	3.41, s	55.2	3.31, s

^a Spectra were recorded at 400 MHz for ¹H NMR and at 101 MHz for ¹³C NMR. ^b Spectra were recorded at 400 MHz for ¹H NMR and at 151 MHz for ¹³C NMR. ^c Spectra exhibited two sets of signals due to C-2′ epimerization of 6.

Penicipyrrolidine T (6) had the molecular formula C₁₆H₂₇NO₄ indicated by HRESIMS, representing an addition of a CH₂O unit relative to 5. The similarity between the ¹H and ¹³C NMR spectra (Table 2) of 6 and those of 5, especially the presence of N-(acetyl)pyrrolidine and 2-hepten-7-yl moiety. The ¹H and ¹³C NMR spectra of 6 showed the presence of a methoxy group (δ_C/H 57.1/3.41). The HMBC correlations from H-2 (δ_H 5.29) to C-3 (δ_C 73.2), C-4 (δ_C 31.1), and C-5 (δ_C 44.6) and from the methoxy group to C-2 confirmed the planar structure of 6 and the presence of the methoxy group in C-2 and the hydroxyl group in C-3 (Figure 2). We expect to establish the relative configuration of 6 by J-based configuration analysis, ROESY [23]. Although the ²J_C-3,H-2 and ²J_C-2,H-3 coupling constants were too small for detection in the HECADE spectra, their minimal values are consistent with, and indeed support, the assigned 2S*,3S* relative configuration of compound 6. Compound 6 also exhibits dual NMR signals due to C-2′ epimerization. However, all attempts to unambiguously determine the configuration of 6 were inconclusive. Furthermore, the limited quantity of isolated compound 6, which was subsequently utilized for biological assays, precluded further analysis. Therefore, the absolute configuration of 6 remains to be elucidated.

Penicipyrrolidine U (7) was isolated as a colorless oil. The molecular formula C₁₃H₂₃NO₂ (three degrees of unsaturation) was established by HRESIMS ([M + H]⁺, calcd for 226.1807) and ¹³C NMR data (Table 2). A detailed analysis of the NMR spectra (Table 2) suggested that 7 possesses the same 1-azaneyl-2-methyldec-8-ene-1,3-dione side chain as 15 [22]. The ¹H-¹H COSY cross peaks identified an ethyl group (C-3–C-2) and HMBC correlations from H-2 to C-1′ established the gross structure as N-ethyl-2-methyl-3-oxodec-8-enamide (Figure 2).

HRESIMS established the molecular formula of penicipyrrolidine V (8), a yellow oil, as C₁₅H₂₃NO₃, which contains one more oxygen atom than that of (–)-penicilactam A (11) [24]. In the ¹H and ¹³C NMR spectra of 8, two methyl groups (δ_C/H 10.2/1.79; 18.1/1.64), two methines (δ_C/H 75.5/4.49) and (δ_C/H 92.4/5.02), two olefinic methines (δ_C/H 131.0/5.40) and (δ_C/H 125.4/5.39) and three quaternary carbons comprising one carbonyl carbon (δ_C 163.4) and two olefinic carbons (δ_C 163.4 and 106.7). Detailed NMR comparison between 8 and 11 established that the sole difference was the conversion of the C-3 methylene group into an oxygenated methine. Key HMBC correlations from H-4 to C-2 and C-3, and from H-5 to C-3 and C-4, along with COSY correlations between H-2, H-3, and H-4, confirmed the proposed change (Figure 2). Further analysis of ¹H-¹H COSY and HMBC spectra confirmed that the remainder of the structure is identical to that of 11 (Figure 2). In order to establish the absolute configuration of C-2 and C-3, TD-DFT calculations were performed on two possible enantiomeric structures of 2S, 3R (8a) and 2S, 3S (8b). The calculated ECD spectrum of 8a was in good agreement with the experimental one for 8 (Figure 3). Thus, the absolute configuration was established as depicted.

Figure 3. Experimental and calculated ECD spectra of compound 8.

HRESIMS established the molecular formulas of penicipyrrolidines W and X (9 and 10) as C₁₅H₂₃NO₂ (m/z 250.1803 [M + H]⁺, calcd for 250.1807) and C₁₅H₂₅NO₂ (m/z 252.1960 [M + H]⁺, calcd for 252.1964), respectively. In contrast to 9, which shares an identical formula with 11, compound 10 contains two more hydrogen atoms than 11, identifying it as a dihydrogenated analogue. The ¹³C NMR data of 9 (Table 3) showed close similarity to those of 11, with the exception of the chemical shifts of three carbons attributable to two olefinic units and one terminal methyl group. Comprehensive analyses of the ¹H-¹H COSY and HMBC spectra established the connectivity from the terminal methyl at C-10′ to the olefinic methine (C-7′) via two methylene units (C-8′ and C-9′), indicating that 9 possessed the double bond at C-6′–C-7′. The E-geometry of this double bond was assigned based on the coupling constant ³J_{H-6′/H-7′} = 15.5 Hz. Analysis of the ¹H and ¹³C NMR data (Table 3) indicated that the data for 10 differed from those of 11 primarily by the absence of signals corresponding to olefinic functionalities, suggesting that 10 features a saturated carbon chain. The absolute configuration of C-2 was established as S by comparing its calculated ECD curve (2S) with the experimental ECD curve of 9 and 10 (Figures S58 and S65).

Table 3. ¹H and ¹³C NMR spectroscopic data for compounds 8–10 in CDCl₃ (δ in ppm, J in Hz).

No.	8 a		9 b		10 a
	δC	δH	δC	δH	δC	δH
2	92.4	5.02, d (3.8)	87.9	5.20, dd (6.0, 4.5)	87.7	5.20, dd (6.0, 4.6)
3	75.5	4.49, m	31.9	2.29, m; 2.21, m	31.9	2.28, m; 2.13, m
4	30.1	2.28, m; 1.93, m	22.1	2.01, m; 1.85, m	22.0	2.00, m; 1.86, m
5	41.8	3.65, m	44.6	3.73, m; 3.42, m	44.4	3.72, ddd (11.2, 7.5, 6.2); 3.42, ddd (11.2, 7.6, 5.9)
1′	163.4		163.7		164.0
2′	106.7		107.0		106.5
3′	163.4		163.2		163.7
4′	30.6	2.30, dt (15.0, 7.8); 2.24, m	31.1	2.36, m; 2.22, m	30.7	2.28, m; 2.17, m
5′	26.4	1.53, p (7.6)	30.1	2.21, m	27.0	1.50, m
6′	29.3	1.38, m	128.8	5.39, m	29.4	1.29, m
7′	32.4	1.99, q (6.4)	131.9	5.45, dt (15.1, 6.5)	29.2	1.29, m
8′	131.0	5.40, m	34.8	1.95, q (7.3)	31.9	1.29, m
9′	125.4	5.39, m	22.8	1.35, m	22.8	1.32, m; 1.26, m
10′	18.1	1.64, d (4.7)	13.9	0.88, t (7.4)	14.2	0.87, t (6.9)
11′	10.2	1.79, s	10.3	1.79, s	10.2	1.79, s

^a Spectra were recorded at 400 MHz for ¹H NMR and at 151 MHz for ¹³C NMR. ^b Spectra were recorded at 600 MHz for ¹H NMR and at 151 MHz for ¹³C NMR.

Five known compounds were identified as (–)-penicilactam A (11), scalusamides A–C (12–14) and N-(2-methyl-3-oxodec-8-enoyl)-2-pyrroline (15), by comparing their physicochemical and spectroscopic data with the reference data [11,24,25,26].

2.2. Glucose Uptake in L6 Skeletal Muscle Cells

The cytotoxicity of compounds 1–10 against L6 cells was assessed using the CCK-8 assay. Compounds 1–9 showed no significant effect on the viability of L6 cells at concentrations up to 100 μM, whereas only compound 10 exhibited mild cytotoxicity at this concentration (Figure S77). Therefore, a safe working concentration of 10 μM was selected for subsequent experiments.

The stimulatory effects of the isolated compounds on glucose uptake were evaluated in L6 skeletal muscle cells using the 2-NBDG assay. At a concentration of 10 μM, the compounds displayed a range of activities. Notably, compound 2 emerged as the most potent, enhancing glucose uptake by 3.83-fold relative to the untreated control. This activity was comparable to that of the positive control, metformin. Compounds 3 and 4 also showed significant, though lesser, effects, increasing uptake by 1.84-fold and 1.25-fold, respectively. These results clearly indicate that compound 2 is a promising candidate for enhancing glucose metabolism.

3. Materials and Methods

3.1. General Experimental Procedures

Optical rotations were determined using a PerkinElmer Model 341 polarimeter (PerkinElmer Inc., Waltham, MA, USA), UV-Vis absorption spectra were obtained with a Shimadzu UV-1700 spectrophotometer (Shimadzu Corporation, Kyoto, Japan), and IR spectra were collected on a Thermo Nexus 470 FT-IR spectrometer (Thermo Electron Corporation, Waltham, MA, USA). NMR spectra were acquired using a Biospin AV 400 NMR instrument (Bruker BioSpin GmbH, Rheinstetten, Germany) at 400 MHz for ¹H NMR and 100 MHz for ¹³C NMR. High-resolution mass spectrometry data were obtained on an LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Column chromatography was performed using silica gel (200–300 mesh, Anhui Liangchen Guiyuan Material Ltd., Liuan, China), and size-exclusion chromatography was conducted with Sephadex LH-20 (Cytiva, Uppsala, Sweden).

3.2. Fungal Material

The fungus Penicillium sp. DM27, derived from mangrove roots, was isolated from the rhizosphere soil of Bruguiera gymnorrhiza (L.) Poir. on 26 September 2013 in the Tachalab subdistrict of the Tamai district in Chantaburi Province, Thailand. Tachalab is located at 102° E 3.4′ longitude, and 12° N 32′ latitude. The fungus was identified according to its morphological characteristics and the sequences of the internal transcribed spacer (ITS). A voucher specimen (Penicillium sp. DM27) is available for inspection at the School of Pharmaceutical Sciences, Wuhan University.

3.3. Mass Culture

Penicillium sp. DM27 was initially inoculated into potato dextrose agar medium, which consisted of 6 g of potato extract, 20 g of glucose, 15 g of sea salt, 20 g of agar, and 1 L of distilled water. The pH of the solution was subsequently adjusted to 6.5, and it was incubated for seven days at 28 °C. Following this incubation period, the culture was transferred to a 300 mL potato dextrose liquid medium contained in a 1 L flask and fermented for 28 days at 28 °C. A total of 60 flasks of the liquid medium were prepared for scale-up in this study.

3.4. Extraction and Isolation

The filtered brown culture yielded mycelia and a liquid phase. The mycelia were ultrasonically extracted with 100% acetone (3 × 3 L), and the combined extracts were concentrated in vacuo to give a crude extract. This extract was then combined with the liquid phase (30 L) and fractionated over macroreticular resin HP20, eluting with MeOH/H₂O (30%, 50%, 70%, 90%) to yield four fractions, designated as A, B, C, and D. Fraction A (5.24 g) was fractionated by silica gel column chromatography (CC) with a stepwise gradient of CH₂Cl₂/MeOH (40:1 to 1:1, v/v) to yield eighteen subfractions (Fr.A.1–Fr.A.18). Fr.A.3 (26 mg) was further purified by RP-HPLC on a Sepex-C18 column (250 × 10 mm, 5 μm) using an isocratic eluent of MeOH/H₂O (60:90, v/v) over 30 min at a flow rate of 3.0 mL/min, affording compound 1 (1 mg, t_R = 20 min). Fr.A.3 (108 mg) was chromatographed over Sephadex LH-20 (eluent: MeOH/CH₂Cl₂, 1:1) to give five subfractions (Fr.A.5.1–Fr.A.5.5). Subsequent purification of Fr.A.5.3 (23 mg) by RP-HPLC with MeOH/H₂O (35:50, v/v; 45 min) yielded compound 4 (8 mg, t_R = 20 min) and 7 (1 mg, t_R = 25 min). Fr.A.18 (98 mg) was separated Sephadex LH-20 washed with MeOH-CH₂Cl₂ = 1:1 to provide five fractions (Fr.A.18.1 to Fr.A.18.5) then Fr.A.18.5 (15 mg) separated by RP-HPLC with MeOH-H₂O (55:45 v/v) to yield 10 (1 mg, t_R = 20 min). The Fr.B (3.20 g) was separated into ten fractions (Fr.B.1 to Fr.B.10) by silica gel CC with elution with mixtures of PE/EA from 10:1 to 0:1 (v/v). Fr.B.9 (23 mg) was further separated into four fractions (Fr.B.9.1 to Fr.B.9.4) with CH₂Cl₂/MeOH 30:1 to 0:1. Fr.B.9.2 was separated by RP-HPLC with MeOH:H₂O (50:50 v/v, 30 min) to yield 3 (2 mg, t_R = 20.4 min). Fr.B.9.3 (25 mg) was further purified by RP-HPLC with MeOH:H₂O (50:30 to 70:20, 20 min) to afford 2 (5 mg, t_R = 15 min). Fr.C (4.50 g) was subjected to silica gel columns with CH₂Cl₂/MeOH (100:1 to 0:1, v/v) to nineteen fractions (Fr.C.1 to Fr.C.19). Fr.C.4 (340 mg) was separated by Sephadex LH-20 eluting with 1:1 CH₂Cl₂–MeOH into four fractions. (Fr.C.4.1 to Fr.C.4.4) and the resulting subfractions Fr.C.4.1 (35 mg) were purified by RP-HPLC to afford 6 (3 mg, t_R = 20 min, 30% MeCN) and 8 (1 mg, t_R = 20 min, 37% MeCN). Fr.C.6 (500 mg) was subjected to Sephadex LH-20 (MeOH-CH₂Cl₂/1:1) and the resulting subfractions were purified by RP-HPLC with 30% MeCN in H₂O for 30 min to afford 9 (1 mg, t_R = 17min). Fr.C.7 (100 mg) was separated Sephadex LH-20 washed with MeOH-CH₂Cl₂/1:1 to provide eight fractions (Fr.C.7.1 to Fr.C.7.8). Fr.C.7.3 (30 mg) was purified by RP-HPLC with MeOH:H₂O (50% to 100%, v/v, 20 min) to afford 5 (1 mg, t_R = 11.0 min).

Penicipyrrolidine O (1): Colorless oil; [α${]}_{\mathrm{D}}^{20}$ −22.0 (c 0.10, MeOH); ¹H and ¹³C NMR data (CDCl₃), see Table 1; HRESIMS m/z 246.1463 [M + Na]⁺ (calcd for C₁₃H₂₁NO₂, 246.1470).

Penicipyrrolidine P (2): White amorphous powder; [α${]}_{\mathrm{D}}^{20}$ −15.0 (c 0.08, MeOH); UV (MeOH) λ_max (log ε) 206 (3.87), 286 (3.03) nm; ECD {MeOH, λ [nm] (Δε), c = 0.1 × 10⁻⁴ M} 216 (−7.00), 202.4 (−7.86), 196.4 (−7.50), 189.8 (−17.73); ¹H and ¹³C NMR data (CDCl₃), see Table 1; HRESIMS m/z 252.1953 [M + H]⁺ (calcd for C₁₅H₂₅NO₂, 252.1964).

Penicipyrrolidine Q (3): Colorless oil; [α${]}_{\mathrm{D}}^{20}$ +18.2 (c 0.02, MeOH); UV (MeOH) λ_max (log ε) 287 (2.01), 224 (2.42), 203 (3.10) nm; ECD {MeOH, λ [nm] (Δε), c = 0.1 × 10⁻⁴ M} 241 (−13.00), 206.9 (−7.25), 191.8 (−8.98); IR (KBr) ν_max 3446, 2926, 1624, 1515, 1384, 1318, 1122, 1053 cm⁻¹; ¹H and ¹³C NMR data (CDCl₃), see Table 1; HRESIMS m/z 290.1718 [M + Na]⁺ (calcd for C₁₅H₂₅NO₃, 290.1732).

Penicipyrrolidine R (4): Colorless oil; [α${]}_{\mathrm{D}}^{20}$ −24.6 (c 0.11, MeOH); UV (MeOH) λ_max (log ε) 203 (4.42), 273 (3.23) nm; ECD {MeOH, λ [nm] (Δε), c = 0.1 × 10⁻⁴ M} 211.6 (+9.47), 198.4 (−17.30), 189 (+42.80); ¹H and ¹³C NMR data (CDCl₃), see Table 1; HRESIMS m/z 288.1563 [M + Na]⁺ (calcd for C₁₅H₂₃NO₃, 288.1576).

Penicipyrrolidine S (5): Colorless oil; [α${]}_{\mathrm{D}}^{20}$ −6.6 (c 0.02, MeOH); ¹H and ¹³C NMR data (CDCl₃), see Table 2; HRESIMS m/z 290.1718 [M + Na]⁺ (calcd for C₁₅H₂₅NO₃, 290.1732).

Penicipyrrolidine T (6): Colorless oil; [α${]}_{\mathrm{D}}^{20}$ −15.3 (c 0.10, MeOH); ¹H and ¹³C NMR data (CDCl₃), see Table 2; HRESIMS m/z 320.1823 [M + Na]⁺ (calcd for C₁₆H₂₇NO₄, 320.1837).

Penicipyrrolidine U (7): Colorless oil; [α${]}_{\mathrm{D}}^{20}$ −18.7 (c 0.10, MeOH); ¹H and ¹³C NMR data (CDCl₃), see Table 2; HRESIMS m/z 226.1800 [M + H]⁺ (calcd for C₁₃H₂₃NO₂, 226.1807).

Penicipyrrolidine V (8): Yellow oil; [α${]}_{\mathrm{D}}^{20}$ −9.3 (c 0.10, MeOH); ¹H and ¹³C NMR data (CDCl₃), see Table 3; HRESIMS m/z 288.1571 [M + Na]⁺ (calcd for C₁₅H₂₃NO₃, 288.1576).

Penicipyrrolidine W (9): Colorless oil; [α${]}_{\mathrm{D}}^{20}$ −2.6 (c 0.10, MeOH); ¹H and ¹³C NMR data (CDCl₃), see Table 3; HRESIMS m/z 250.1803 [M + H]⁺ (calcd for C₁₅H₂₃NO₂, 250.1807).

Penicipyrrolidine X (10): Colorless oil; [α${]}_{\mathrm{D}}^{20}$ −2.0 (c 0.10, MeOH); ¹H and ¹³C NMR data (CDCl₃), see Table 3; HRESIMS m/z 252.1960 [M + H]⁺ (calcd for C₁₅H₂₅NO₂, 252.1964).

3.5. Computational Analysis

To achieve conformational optimization of the stereoisomers, computational studies were conducted using density functional theory (DFT). Initial conformational sampling was performed with the MMFF94 molecular mechanics force field. The resulting low-energy conformers were subsequently optimized at the DFT level using the Gaussian 09 software package with the 6-311G (2d, p) basis set for all atoms [27]. Room-temperature equilibrium populations were estimated based on the Boltzmann distribution law (Equation (1)). The ECD spectra for individual conformers were simulated using a Gaussian function, and the final composite spectra were generated by Boltzmann weighted averaging of the respective conformer spectra.

$${\displaystyle \frac{N_i}{N}}={\displaystyle \frac{g_i{e}^{-\frac{E_i}{k_{\mathrm{B}}T}}}{{\displaystyle \sum g_i{e}^{-\frac{E_i}{k_{\mathrm{B}}T}}}}}$$

(1)

where N_i is the number of conformer i with energy E_i and degeneracy g_i at temperature T, and k_B is Boltzmann constant.

3.6. Cell Culture and Treatment

Rat skeletal muscle L6 cells were obtained from Wuhan Procell Life Science & Technology Co., Ltd., Wuhan, China and were cultured in complete medium consisting of 89% α-MEM, 10% FBS, and 1% antibiotics. After passaging and stable growth, the cells were cultured in α-MEM medium supplemented with 2% FBS. The medium was changed daily for 7 days to induce differentiation into myotubes.

The potential cytotoxicity of the test compounds was evaluated using a Cell Counting Kit-8 (CCK-8) assay. Briefly, L6 myotubes were seeded in 96-well plates at a density of 1 × 10⁴ to 5 × 10⁴ cells per well. After differentiation, the cells were treated with the test compounds at four different concentrations (3, 10, 30, and 100 μM), prepared in a medium containing a final concentration of 0.1% DMSO. A vehicle control group (0.1% DMSO in medium) and a blank control (medium only) were included. After 24 h of incubation, the medium was replaced with 100 μL of fresh α-MEM containing 10% (v/v) CCK-8 reagent. The plates were incubated for an additional 2 h at 37 °C. The absorbance was measured at 450 nm using a microplate reader. Cell viability was expressed as a percentage relative to the vehicle control group.

Myotubes were seeded in 96-well plates at a density of 1 × 10⁴ to 5 × 10⁴ cells per well, with 100 μL of α-MEM medium added to each well until the cells covered the dish. Three groups were established: a blank control, a metformin positive control, and a drug administration group (10 μM), each with three sub-wells. 100 μL of α-MEM medium containing 2-NBDG and each compound (10 μM) was added to the myotubes and incubated for 30 min in a constant temperature incubator. After incubation, the 96-well plates were centrifuged for 5 min at 400× g, the supernatant was discarded, and 200 L of kit buffer was added to each cell well, mixed, and centrifuged again at 400× g for 5 min at room temperature. This process was repeated. The glucose uptake capacity of the cells was assessed by measuring the 2-NBDG fluorescence intensity.

4. Conclusions

The isolation of penicipyrrolidines O–X (1–10) from the mangrove-derived fungus Penicillium sp. DM27 expands the structural diversity of pyrrolidine derivatives characterized by eight- or eleven-carbon atom side chains. In compounds 2–4 and 15, the two sets of signals are ascribed to rotamers arising from restricted amide bond rotation, which exist in a dynamic equilibrium, as supported by the observation of trans and cis amide conformers in a ratio of approximately 3:1. The rotational isomers are absent in compounds 1, 5–6, and 12–14 due to the bulky substituent at the 2-position. The two sets of signals observed in compounds 6 and 12–14 originates from epimerization at the 2′-position, a configurational inversion process at a chiral center. Immediate analysis of the freshly isolated pure compound—as exemplified by 12 (Figures S69 and S70)—reveals a single set of NMR signals. However, upon prolonged standing, epimerization proceeds, leading to the reappearance of two sets of signals, as illustrated by 13 and 14 (Figures S71–S74). Consequently, the presence of rotational isomerism alongside time-dependent epimerization at C-2′ precluded the reliable determination of the absolute configurations for those compounds using methods such as the modified Mosher′s method. Upon evaluation of glucose uptake activity in L6 cells for all compounds, compound 2 demonstrated the highest activity at 3.83-fold, comparable to metformin, while compounds 3 and 4 were less active at 1.84-fold and 1.25-fold, respectively.